miR172 Regulates WUS during Somatic Embryogenesis in Arabidopsis via AP2.

In plants, the embryogenic transition of somatic cells requires the reprogramming of the cell transcriptome, which is under the control of genetic and epigenetic factors. Correspondingly, the extensive modulation of genes encoding transcription factors and miRNAs has been indicated as controlling the induction of somatic embryogenesis in Arabidopsis and other plants. Among the MIRNAs that have a differential expression during somatic embryogenesis, members of the MIRNA172 gene family have been identified, which implies a role of miR172 in controlling the embryogenic transition in Arabidopsis. In the present study, we found a disturbed expression of both MIRNA172 and candidate miR172-target genes, including AP2, TOE1, TOE2, TOE3, SMZ and SNZ, that negatively affected the embryogenic response of transgenic explants. Next, we examined the role of AP2 in the miR172-mediated mechanism that controls the embryogenic response. We found some evidence that by controlling AP2, miR172 might repress the WUS that has an important function in embryogenic induction. We showed that the mechanism of the miR172-AP2-controlled repression of WUS involves histone acetylation. We observed the upregulation of the WUS transcripts in an embryogenic culture that was overexpressing AP2 and treated with trichostatin A (TSA), which is an inhibitor of HDAC histone deacetylases. The increased expression of the WUS gene in the embryogenic culture of the hdac mutants further confirmed the role of histone acetylation in WUS control during somatic embryogenesis. A chromatin-immunoprecipitation analysis provided evidence about the contribution of HDA6/19-mediated histone deacetylation to AP2-controlled WUS repression during embryogenic induction. The upstream regulatory elements of the miR172-AP2-WUS pathway might involve the miR156-controlled SPL9/SPL10, which control the level of mature miR172 in an embryogenic culture.

Enhancing In Vitro Production of the Tree Fern Cyathea delgadii and Modifying Secondary Metabolite Profiles by LED Lighting.

The tree ferns are an important component of tropical forests. In view of this, the enhancement of in vitro production of these plants is needed. Thus, the effect of different light-emitting diodes (LEDs) as well as control fluorescent lamps (Fl) and a 3-week-long period of darkness at the beginning of in vitro culture on micropropagation of the tree fern Cyathea delgadii Sternb. was analysed. Moreover, the photosynthetic pigment content and secondary metabolite profiles were estimated. The period of darkness contributed to a high production of somatic embryo-derived sporophytes and a low production of gametophytes. The formation of new sporophytes was stimulated by RBY (35% red, 15% blue, and 50% yellow) and B (100% blue) lights when the stipe explants or whole young sporophytes were used in the culture, respectively. The elongation of the roots and leaves was stimulated by RBfR light (35% red, 15% blue, and 50% far red), while root production increased under RBY light. The RB (70% red and 30% blue) and B lights stimulated the accumulation of chlorophyll better than Fl light. The most abundant metabolite found in the plant extracts was trans-5-O-caffeoylquinic acid (1.013 µg/mg of dry weight). The extract obtained from plants growing in a greenhouse had the best antioxidant activity.

The Origin of the Cyathea delgadii Sternb. Somatic Embryos Is Determined by the Developmental State of Donor Tissue and Mutual Balance of Selected Metabolites.

Somatic embryogenesis is the formation of a plant embryo from a cell other than the product of gametic fusion. The need to recognize the determinants of somatic cell fate has prompted investigations on how endogenous factors of donor tissues can determine the pattern of somatic embryo origin. The undertaking of this study was enabled by the newly developed experimental system of somatic embryogenesis of the tree fern Cyathea delgadii Sternb., in which the embryos are produced in hormone-free medium. The contents of 89 endogenous compounds (such as sugars, auxins, cytokinins, gibberellins, stress-related hormones, phenolic acids, polyamines, and amino acids) and cytomorphological features were compared between two types of explants giving rise to somatic embryos of unicellular or multicellular origin. We found that a large content of maltose, 1-kestose, abscisic acid, biologically active gibberellins, and phenolic acids was characteristic for single-cell somatic embryo formation pattern. In contrast, high levels of starch, callose, kinetin riboside, arginine, and ethylene promoted their multicellular origin. Networks for visualization of the relations between studied compounds were constructed based on the data obtained from analyses of a Pearson correlation coefficient heatmap. Our findings present for the first time detailed features of donor tissue that can play an important role in the somatic-to-embryogenic transition and the somatic embryo origin.

Somatic embryogenesis: from discovery through investigation to application / Somatyczna embriogeneza: od odkrycia przez badania do praktycznego wykorzystania.

Plant cells possess the remarkable ability to adapt to environmental changes. It is manifested by formation of embryos directly from the cells of plant body, bypassing the fertilization stage. These embryo structures develop into complete plants. The process itself, to distinguish the path of formation and emphasize consistency with zygotic embryogenesis, is referred to as somatic embryogenesis (SE). Although more than 60 years have passed since the first publication on the phenomenon has been written, the mechanism of reprogramming of a somatic cell into an embryogenic one is still not fully understood. This is a critical step in SE that can be induced by exo- and endogenous factors and stress treatments. The exposition of plant material to these factors affects the reorganization of the chromatin structure and gene expression, which can consequently trigger the program of embryogenesis. The paper reviews current knowledge on how the identity of totipotent cells is determined and the which stimuli are required to reprogram somatic cell development. Knowledge of key molecular regulators and the network of relationships that control the SE induction is summarized. Issues that are important for enhancing the understanding of the mechanisms underlying totipotency are also defined. Finally, the practical potential of SE is demonstrated, and examples of its use are provided.

Symplasmic Isolation Contributes to Somatic Embryo Induction and Development in the Tree Fern Cyathea delgadii Sternb.

In this report, we describe studies on symplasmic communication and cellular rearrangement during direct somatic embryogenesis (SE) in the tree fern Cyathea delgadii. We analyzed changes in the symplasmic transport of low-molecular-weight fluorochromes, such as 8-hydroxypyrene-1,3,6-trisulfonic acid, trisodium salt (HPTS) and fluorescein (delivered to cells as fluorescein diacetate, FDA), within stipe explants and somatic embryos originating from single epidermal cells and developing during 16-d long culture. Induction of SE is preceded by a restriction in fluorochrome distribution between certain explant cells. Microscopic analysis showed a series of cellular changes like a decrease in vacuole size, increase in vacuole numbers, and increased density of cytoplasm and deposition of electron-dense material in cell walls that may be related with embryogenic transition. In somatic embryos, the limited symplasmic communication between cells was observed first in linear tri-cellular embryos. Further development of the fern embryo was associated with the formation of symplasmic domains corresponding to the four segments of the plant body. Using symplasmic tracers, we provided evidence that the changes in plasmodesmata permeability are corelated with somatic-to-embryogenic transition and somatic embryo development.

Proteomic analysis of stipe explants reveals differentially expressed proteins involved in early direct somatic embryogenesis of the tree fern Cyathea delgadii Sternb.

Using cyto-morphological analysis of somatic embryogenesis (SE) in the tree fern Cyathea delgadii as a guide, we performed a comparative proteomic analysis in stipe explants undergoing direct SE. Plant material was cultured on hormone-free medium supplemented with 2% sucrose. Phenol extracted proteins were separated using two-dimensional gel electrophoresis (2-DE) and mass spectrometry was performed for protein identification. A total number of 114 differentially regulated proteins was identified during early SE, i.e. when the first cell divisions started and several-cell pro-embryos were formed. Proteins were assigned to seven functional categories: carbohydrate metabolism, protein metabolism, cell organization, defense and stress responses, amino acid metabolism, purine metabolism, and fatty acid metabolism. Carbohydrate and protein metabolism were found to be the most sensitive SE functions with the greatest number of alterations in the intensity of spots in gel. Differences, especially in non-enzymatic and structural protein abundance, are indicative for cell organization, including cytoskeleton rearrangement and changes in cell wall components. The highest induced changes concern those enzymes related to fatty acid metabolism. Global analysis of the proteome reveals several proteins that can represent markers for the first 16days of SE induction and expression in fern. The findings of this research improve the understanding of molecular processes involved in direct SE in C. delgadii.